ADVANCING GENE-EDITING NUCLEASES FOR DIVERSE ZEBRAFISH APPLICATIONS

推进基因编辑核酸酶在多种斑马鱼中的应用

基本信息

批准号：
10737505
负责人：
RANDALL T PETERSON
金额：
$ 49.28万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-20 至 2027-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10737505
关键词：
Address Animals Area Bar Codes Biomedical Research Biomedical Technology CRISPR screen CRISPR/Cas technology Cells Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Collection Communities DNA Development Disease model Embryo Engineering Financial Support Fishes Funding Future Gene Transfer Techniques Genes Genetic Diseases Genome Genomics Integrase Location Locomotion Methods Microfluidics Modification Molecular Monitor Morphology Mutation Needles Organism Phenotype Plasmids Proteins Recovery Regulator Genes Regulatory Element Research Personnel Site Specific qualifier value Specificity System Technology Transgenic Organisms Visual Zebrafish cell type combinatorial gene function genome editing genomic locus high throughput screening high throughput technology innovation interest metabolomics molecular phenotype mutant nanolitre new technology nuclease prime editing promoter recombinase reverse genetics screening single-cell RNA sequencing small molecule technology development technology platform tool transcription activator-like effector nucleases

项目摘要

Summary CRISPR-Cas9 and related technologies have dramatically transformed our ability to manipulate the genomes of countless organisms, generate disease models, rapidly discover the functions of genes, and create new treatments for genetic disorders. Nevertheless, the full potential of the field has yet to be realized. Over the past fifteen years, the Peterson and Yeh labs have collaborated in the development of genome editing tools and have contributed to several key advances, including the first use of CRISPR-Cas9 to modify the genome of any animal, the first use of prime editing in zebrafish, and the first technology for high- throughput CRISPR screening in a vertebrate. During the current funding period, we have successfully completed all three of our aims and produced new technologies, TICIT and MIC-Drop, that raise exciting new opportunities for further development and application. In this competitive renewal application, we propose to expand on TICIT and MIC-Drop, developing several useful new gene-editing approaches. Aim 1 builds upon Targeted Integration by CRISPR-Cas9 and Integrase Technologies (TICIT), which utilizes the site-specific DNA recombinase – phiC31 integrase – to insert DNA into genomic target sites that have been pre-specified by CRISPR-Cas9 modification. Unlike traditional transgenic methods which lead to random genome insertion, TICIT enables precise integration of plasmids into the genome at prespecified loci, avoiding inadvertent gene disruption and positional effects. Building upon TICIT, we propose now to expand the utility of integrases for site-specific genome editing. In the renewal application, we propose new technologies to make TICIT more efficient and permit combinatorial uses of multiple integrases in various safe- harbor genomic loci. Additionally, we will use the optimized TICIT platform to develop high-throughput screening systems for isolating synthetic promoters with enhanced cell-state or cell-type specificity. Aim 2 builds upon Multiplexed, Intermixed CRISPR Droplets (MIC-Drop), which combines multiplexed CRISPR sgRNAs, Cas9 protein, and DNA barcodes into nanoliter-volume microfluidic droplets that can then be injected from a single needle into thousands of zebrafish. MIC-Drop enables rapid and efficient disruption of hundreds or thousands of genes, and after identification of phenotypes of interest, the causative gene can be quickly identified by barcode recovery. Building upon MIC-Drop, we propose now to expand the utility of MIC-Drop for molecular phenotyping. Previously, we have used visual inspection to identify zebrafish mutants with interesting phenotypes, followed by PCR-based recovery of the DNA barcodes. We now plan to combine MIC-Drop technology with powerful molecular phenotyping tools, such as single-cell RNA sequencing (scRNAseq) and metabolomics. In this aim, we will develop new methods, barcodes, and workflows that will enable efficient gene disruption and molecular phenotyping at unprecedented scales.

概括 CRISPR-Cas9 和相关技术极大地改变了我们操纵细胞的能力无数生物体的基因组，生成疾病模型，快速发现基因的功能，以及然而，该领域的全部潜力尚未得到充分发挥。在过去的十五年里，Peterson 和 Yeh 实验室在基因组开发方面进行了合作编辑工具，并为多项关键进展做出了贡献，包括首次使用 CRISPR-Cas9 来修饰任何动物的基因组，首次在斑马鱼中使用原代编辑，以及第一个高科技在当前的资助期内，我们已经成功地在脊椎动物中进行了 CRISPR 筛选。完成了我们的所有三个目标并产生了新技术 TICIT 和 MIC-Drop，这引发了令人兴奋的新技术进一步开发和应用的机会在这个竞争性的更新应用中，我们建议：扩展 TICIT 和 MIC-Drop，开发几种有用的新基因编辑方法。目标 1 以 CRISPR-Cas9 和 Integrase Technologies (TICIT) 的靶向整合为基础，利用位点特异性 DNA 重组酶（phiC31 整合酶）将 DNA 插入基因组目标位点，与传统的转基因方法不同，它是通过 CRISPR-Cas9 修饰预先指定的。随机基因组插入，TICIT 能够将质粒精确整合到基因组中预先指定的位点，避免无意的基因破坏和位置效应在 TICIT 的基础上，我们现在建议进行扩展。在更新应用中，我们提出了新的整合酶在位点特异性基因组编辑中的实用性。技术使 TICIT 在各种安全领域更有效地组合使用多个集成此外，我们将使用优化的TICIT平台来开发高通量。用于分离具有增强的细胞状态或细胞类型特异性的合成启动子的筛选系统。 Aim 2 以多重、混合 CRISPR 液滴 (MIC-Drop) 为基础，结合了多重、混合的 CRISPR 液滴 (MIC-Drop) 将 CRISPR sgRNA、Cas9 蛋白和 DNA 条形码转化为纳升体积的微流体液滴，然后可以将其从一根针头注射到数千只斑马鱼中，可以快速有效地破坏细胞。数百或数千个基因，在鉴定出感兴趣的表型后，致病基因可以是在 MIC-Drop 的基础上，我们现在建议扩展 MIC-Drop 的实用性。用于分子表型分析的 MIC-Drop 之前，我们使用目视检查来识别斑马鱼。具有有趣表型的突变体，然后基于 PCR 恢复 DNA 条形码。将 MIC-Drop 技术与强大的分子表型分析工具（例如单细胞 RNA 测序）相结合（scRNAseq）和代谢组学为了实现这一目标，我们将开发新的方法、条形码和工作流程。能够以前所未有的规模进行有效的基因破坏和分子表型分析。